Modified Method for Competitive Reverse Transcription Polymerase Chain Reaction for Rapid and Automated Quantitation...

Background: Competitive reverse transcription polymerase chain reaction (RT-PCR) has been used increasingly to quantitate messenger RNA (mRNA) levels; however, conventional competitive RT-PCR methods require four or five reactions per sample of RNA, employing serial dilutions of an internal competitor sequence, making analysis of multiple samples of tedious process. A modified method is described by which multiple samples and multiple RNA transcripts can be analyzed easily by an automated process. Methods and Results: Transforming growth factor beta-1 (TGF-beta-1) mRNA was assayed in total RNA extracted from cultured human skin fibroblasts. A standard solution of total RNA was first prepared by pooling RNA from several cell lines and stored in aliquots. A 270-bp competitor RNA molecule (RNA mimic) was prepared by in vitro transcription and was added to each reaction. PCR was performed with a fluorescent dye (Hex; Applied Biosystems, Foster City, CA)-labeled sense primer to amplify a 161-bp-long c DNA product of target TGF-beta-1 mRNA sequence and the RNA mimic. The PCR products were analyzed with an automated laser-scanned gel electrophoresis system and the area under the curve (AUC) was used for quantitation. The concentration TGF-beta-1 mRNA in standard RNA was determined by conventional competitive RT-PCR. Subsequently, equal amounts of RNA mimic were mixed with four serial dilutions of standard RNA and 0.1 µg of sample total RNA for RT-PCR. A standard curve was generated using the known dilutions of a standard target RNA solution and ratio of AUC for target to that for mimic for each dilution. The unknown sample was then quantitated by interpolation of its area under the curve ratio on the standard curve. This method had inter- and intra-assay coefficients of variation of less than 10%. Conclusions: This modification is highly reproducible for quantitation of mRNA and significantly reduces the number of PCR reactions required for each assay. It can be used to assay several RNA molecules in a given sample by designing RNA mimics and PCR primers to generate PCR products of different lengths so that they can be analyzed by the laser scanning of a single lane of electrophoretic gel.